Type 1 diabetes (T1D) is a debilitating autoimmune disease with the incidence on the increase in developed countries, suggesting that changes in environment (including changes in microbial environment) may be responsible. We have pioneered and still continue the studies to delineate the role of innate immune receptors in initiation and prevention of T1D. We and many other researchers involved in microbiota studies are faced with the same issue - how to explain the effects of host genetics interacting with microbiota. Our results acquired during the grant period now show that multiple unrelated bacterial lineages can protect MyD88 KO mice from T1D. That makes us focus on identification of important tolerizing receptors rather then on specific microbial lineages, as we proposed earlier. We have found that MyD88-negative B cells provided a strong degree of protection. We came up with a novel hypothesis that B cells allow Teff cells to avoid suppression by Tregs. The role of the microbiota and MyD88 in that anti-suppression needs to be uncovered. We found that signaling adaptor TRIF was also instrumental in protection of MyD88KO mice (we do not know whether there is a connection to B cells yet). Moreover, in the recent years new incredible facts concerning the microbiota came to life including participation of microbiota in viral infections [refs]. We cannot ignore the fact that an ecotropic murine leukemia virus (MuLV) is upregulated in MyD88 KO NOD mice. Does the combination of a retrovirus and bacteria block T1D development? Finally, we were convinced that microbes are not required for T1D development (as in GF NOD mice), but we now have an indication that T1D-inducing microbes exist, L.murinus being one of them. We will further investigate the pro-diabetic properties of microbes including alleged facilitators of the human T1D. It is a complex proposal, but underlying and unifying theme is the role of intestinal microbiota in T1D development and prevention. The new Specific Aims are:. 1. Cellular basis for microbiota-dependent control of autoimmunity. We will follow-up on the role of B cells as protectors in MyD88-negative NOD mice, and test the combined input of B cells, dendritic cells and monocytes in the protection; 2. The role of TRIF in protective effect in T1D. We will find the cellular basis and signaling receptors that participate in microbially-triggered tolerization signals; 3. Test the role of microbes in the newly proposed role for B cells in T1D. We will test the new hypothesis that B cells make Teff cells insensitive to suppression by Tregs and investigate the role of microbiota and MyD88 signaling in the process; 4. Determine the role of pro-diabetic microbiota taking advantage of NOD mice with genetic defect in NADPH- oxidase.